The goal of this research is to elucidate metabolic pathways in both the intra- and extracellular regime. In this application we will develop fluorescent probes capable of determining the concentrations of a wide range of hydrophobic metabolites in both extra and intracellular fluids. Probes will be constructed by fluorescent labeling mutants of fatty acid binding proteins (FABP). A unique feature of these probes is they enable the measurement of the concentration of unbound (aqueous monomers) hydrophobic metabolites, thereby providing direct measurements of the molecular constituents of the metabolic pathways. The focus of this work will be the development of probes for determining the concentration of unbound free fatty acids (FFAu). In health, circulating FFA are highly regulated as is the distribution (profile) of the as many as 40 different species of FFA. FFA serve a variety of essential functions; they provide a major portion of the body's energy and are important as signaling molecules. Even modest changes in the levels and distributions of FFA that may occur in diseases such as diabetes, cardiac and cerebral ischemia and probably cancer, can be highly deleterious to human health. Because of technical limitations accurate monitoring of FFA profiles has been very challenging. FFA as well as most hydrophobic metabolites mediates their activities through the unbound (aqueous phase) molecules. However, with the exception of the probes we are developing for FFAu there are virtually no methods for the accurate determination of the unbound molecules in extracellular as well as intracellular fluids. In these studies we will: 1) generate a suite of 40 probes specific to individual FFAu using a high throughput system (HTS) for generating and screening new probes that in preliminary studies has yielded probes with FFA binding profiles that are significantly different from those of ADI FAB, 2) develop methods to determine the concentration of individual FFAu in extracellular fluids containing mixtures of FFAu and 3) develop methods to determine intracellular concentrations of different FFAu in living cells.